Bacterial RNA polymerase is both a central target in mechanistic studies of gene regulation and a highly attractive target for new antibiotic development. Both mechanistic studies and antibiotic development will be enabled by identification and use of new RNA polymerase inhibitors. Because the surface structure and the regulatory properties of RNA polymerase as well as the need for specific antibiotics vary among different bacterial lineages, discovery of lineage-specific inhibitors is a particularly attractive goal. These lineage- specific inhibitors will provide powerful new molecular probes for investigating the biological function of RNA polymerase and valuable lead compounds to replenish the antibiotic development pipeline. The utility of inhibitors for dissecting the complex enzymatic mechanism of RNA polymerase is well documented by recent studies that correlate inhibitor-RNA polymerase co-crystal structures with effects of inhibitors on steps in the mechanism. New inhibitors, especially inhibitors that affect easily crystallized Thermus aquaticus RNA polymerase, will be especially valuable for these studies. The need for new antibiotics is clear. The United States and the world now confront a crisis in antibiotic development. Hospitals have become breeding grounds for opportunistic pathogens, leading to more than 90,000 deaths/yr in the US alone. Methicillin-resistant Staphylococcus aureus (MRSA) is already spreading rapidly outside hospitals in the US and globally, and veterans of the Iraq war are returning with untreatable infections of Acinetobacter baumannii. RNA polymerase is a proven drug target and must be fully exploited in the quest for new antibiotics. Lineage-specific RNA polymerase inhibitors are excellent lead compounds for development of new antibiotics targeting the bacteria that pose the greatest threats to human health. We will develop a novel high-throughput screen for lineage-specific RNA polymerase inhibitors using specialized bioreporter bacteria that detect the activity of recombinant bacterial RNA polymerases in situ using a fluorescent signal. The bioreporter assay already is working, is ideally adaptable for high-throughput screening, and has the important advantage of identifying inhibitors that can enter and work within a bacterial cell. The specific aims are to (1) establish a high-throughput screen of inhibitors of B. anthracis and T. aquaticus RNA polymerase; (2) validate robust and reproducible behavior of the high-throughput assay using known RNA polymerase inhibitors; (3) perform an initial screen using compound libraries available at UW Madison and then supply the assay to NIH-funded high-throughput screening facilities; and (4) extend the high-throughput assay to other high priority targets for antibiotic development.